GRK5 is a regulator of fibroblast activation and cardiac fibrosis.

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.

Perturbation of the interactions of calmodulin with GRK5 using a natural product chemical probe.

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.

A peptide of the N terminus of GRK5 attenuates pressure-overload hypertrophy and heart failure.

Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase 5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca2+-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.

New Insights in Cardiac ß-Adrenergic Signaling During Heart Failure and Aging.

Heart failure (HF) has become increasingly common within the elderly population, decreasing their survival and overall quality of life. In fact, despite the improvements in treatment, many elderly people suffer from cardiac dysfunction (HF, valvular diseases, arrhythmias or hypertension-induced cardiac hypertrophy) that are much more common in an older fragile heart. Since ß-adrenergic receptor (ß-AR) signaling is abnormal in failing as well as aged hearts, this pathway is an effective diagnostic and therapeutic target. Both HF and aging are characterized by activation/hyperactivity of various neurohormonal pathways, the most important of which is the sympathetic nervous system (SNS). SNS hyperactivity is initially a compensatory mechanism to stimulate contractility and maintain cardiac output. Unfortunately, this chronic stimulation becomes detrimental and causes decreased cardiac function as well as reduced inotropic reserve due to a decrease in cardiac ß-ARs responsiveness. Therapies which (e.g., ß-blockers and physical activity) restore ß-ARs responsiveness can ameliorate cardiac performance and outcomes during HF, particularly in older patients. In this review, we will discuss physiological ß-adrenergic signaling and its alterations in both HF and aging as well as the potential clinical application of targeting ß-adrenergic signaling in these disease processes.

Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases.

Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels.

A repressor protein, Mnt, is a novel negative regulator of vascular smooth muscle cell hypertrophy by angiotensin II and neointimal hyperplasia by arterial injury.

OBJECTIVE: The Max-interacting protein Mnt is a transcriptional repressor that can antagonize the transcriptional and proliferation-related activities of Myc. Here, we tested the hypothesis that Mnt is a negative regulator of pathological vascular remodeling. METHODS: Adenovirus encoding Mnt or control GFP was infected to cultured rat vascular smooth muscle cells (VSMC) and carotid arteries after a balloon angioplasty. RESULTS: In VSMC, adenoviral gene transfer of Mnt suppressed angiotensin II-induced protein expression of early growth response protein-1 (Egr1) and its promoter activation. Mnt adenovirus did not interfere with upstream signaling of angiotensin II. Angiotensin II-induced protein accumulation in VSMC was inhibited by Mnt adenovirus. Mnt adenovirus also inhibited platelet-derived growth factor-induced VSMC proliferation. Moreover, Mnt adenovirus prevented neointima formation in response to arterial injury. The adenoviral Mnt gene transfer also prevented Egr1 induction in neointima. CONCLUSION: These data identify Mnt as a previously unrecognized negative regulator of pathological vascular remodeling.